Wang Y et al / Acta Pharmacol Sin 2003 Dec; 24 (12): 1281-1286

Increase of ₁-adrenergic receptor gene expression induced by nicotine in hippocampal slice of rat¹

WANG Yong, ZHU Xiao-Nan, YAN Jie², YU Jian-Ping, HUANG Xiao-Hui, CHEN Ru-Zhu³

Department of Pharmacology, Sun Yat-sen University of Medical Science, Guangzhou 510089, China

¹ Supported by Ministry of Public Health Foundation. No 98-1-086.

² Now in Department of Statistics, the 3ed Military Medical University.

³ Correspondence to Prof CHEN Ru-Zhu. Phn 86-20-8733-0561. Fax 86-20-8733-0561. E-mail 196673@163.com

Received 2002-12-23 Accepted 2003-11-03

KEY WORDS nicotine; -adrenergic receptors; hippocampus; reverse transcriptase polymerase chain reaction; Western blotting; radioligand assay

ABSTRACT

AIM: To investigate the effect of nicotine on ₁-adrenergic receptor (₁-AR) in the hippocampal slice of rat. METHODS: Hippocampal slices (400 µm thick) were incubated in artificial cerebrospinal fluid (ACSF) previously saturated with 95 % O₂ and 5 % CO₂ at 28 ºC for 120 min, and then incubated with nicotine 10 µmol/L for 30, 60, 90, and 120 min. mRNA of the ₁-adrenergic receptor was examined with semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), and the protein level was measured by Western blot and RIA. RESULTS: The mRNA gene expression and the protein level of ₁-adrenergic receptor in hippocampal slices were increased after nicotine treatment. The peak of protein occurred later but higher than that of mRNA level. CONCLUSION: Both expression of 1- adrenergic receptor gene transcription and post-transcriptional protein level in rat hippocampus were altered by nicotine.

INTRODUCTION

The effect of nicotine on learning and memory function has been focused recently, some observation from animal and human being behavior experiments suggested that nicotine could improve animal and human being learning and memory^[1-3]. Long-term potentiation (LTP) as the cellular model for learning and memory has been always used to explore the molecular and cellular mechanisms of the cognitive function. LTP involves a number of different neurotransmitter systems, and adrenergic system plays important role in learning and memory^[4,5]. Norepinephrine (NE) is one of these major neuromodulators involved in the induction of LTP; activation of -adrenergic receptor (AR) on synapse enhances LTP strongly^[6], and -AR blocker could block the LTP elicited by the titanic stimulation in the hippocampal slice of rat or induce distribution of learning and memory function in mice^[7], which indicated that norepinephrine (NE) and -adrenergic receptors in the CNS were involved in the learning and memory. It has been confirmed that nicotine can stimulate release of NE^[8,9], facilitate or induce LTP in the hippocampal slice of rat^{[10,11 ]}. LTP induced by nicotine with different parameters of electric stimulation could be blocked with proporanolol, a nonselective -AR antagonist^[7], so adrenergic receptor signaling is important in LTP induced by nicotine^[12]. However, the molecular mechanisms responsible for the effects of that nicotine facilitate LTP induction via ₁ adrenergic receptors are unknown, the present experiment aimed to investigate whether nicotine influences the gene expression of ₁ adrenergic receptors.

MATERIALS AND METHODS

Reagents and animals Nicotine was obtained from Sigma. Antibody for ₁-AR was a product of Santa Cruz Biotechnology Inc. Tripure isolation reagent, PVDF membrane and Lumi-Light^plus Western blotting Substrate Kit were purchased from Rohe. [³H]DHA was from Amersham Pharmacia Biotech Ltd. Sprague-Dawley rats were from the Experimental Animal Center of Sun Yat-sen University (100-120 g, Grade II, Certificate No 26-001 conferred by Medical Animal Management Committee, Guangdong Province). Other chemicals from Sigma were of analytic grade.

Preparation of hippocampal slices Hippocampal slices(400 µm thick) used in the experiment were obtained from Sprague-Dawley rats. Animals were anesthetized with diethyl ether and then the brains were removed rapidly and placed in cold artificial cerebrospinal fluid (ACSF) consisting of (in mmol/L: NaCl 124, KCl 3.4, MgSO₄·7H₂O 1.7, NaHCO₃ 25, CaCl₂ 1.8, glucose 10; pH 7.4) and previously saturated with 95 % O₂ and 5 % CO₂. The hippocampal slices were trans-versed in ACSF and incubated at 28 ºC for 120 min, and then nicotine 10 µmol/L was added. After incubation for 30, 60, 90, and 120 min respectively, the samples were determined.

RNA preparation According to the protocol of Tripure^TM isolation reagent, hippocampal slice with Tripure isolation reagent was homogenized, and then centrifuged at 12000×g for 10 min at 4 ºC. The supernatant was transferred and incubated for 5 min followed by incubation with chloroform for 2-15 min at room temperature. After isolation of the colorless upper aqueous phase isopropanol was added, and the sample was centrifuged again. The pellet was washed with 75 % ethanol.

Determination of the level of ₁-AR mRNA by RT-PCR The level of ₁-AR mRNA was detected by RT-PCR based on the method of Miyahara et al^[13]. The cDNA was synthesized with MluV reverse transcripase. Reaction mixture 20 µL containing 0.2-3.2 µg sample RNA, RNasin ribonuclease, oligo d (T)16 primer, dNTP 1 mmol/L was incubated in RT buffer at 37 ºC for 5 min, then with 200 U MluV reverse transcripase, at 42 ºC for 60 min. The reaction was stopped by heating at 72 ºC for 10 min and immediately chilled on ice. The primers were designed according to software and synthesized by Sangon. The sense primer was 5'-CAT CAC GCT GCC CTT TCG CTA-3', and the antisense primer 5'-CGG TTG GTG ACG AAA TCG C -3'. RT reaction mixture 3 µL was then diluted with PCR buffer containing 0.5 µg of sense primer and antisense primer, dNTP 0.2 mmol/L, and 1 U Tag DNA polymerase. Amplified reaction was performed with a thermocycler for a single 5-min heating step at 95 ºC followed 28 cycles under the conditions: 94 ºC (60 s), 55 ºC (60 s), 72 ºC (60 s); and final extension at 72 ºC for 10 min. The amplification resulted in an expected product of 188 bp. Initially, the number of cycles was titrated for sufficient but still exponential amplification. Aliquot of 8 µL was removed from PCR mixture after different cycles of amplification and were electrophoresed on a 1.5 % agarose gel containing ethidium bromide in TAE 40 mmol/L. Amplified DNA band was scanned and the relative density was quantified. Glyceraldhyde-3-phosphate dehydrogenase (GAPDH) as internal standard was simultaneously amplified using 5'-TCA CCA TCT TCC AGG AGC GAG A-3' as a sense primer and 5'-ATG AGC CCT TCC ACG ATG C-3' as an antisense primer. The amplified fragment of GAPDH was 303 bp.

Determination of ₁-AR protein level by Western blot analysis Western blot analysis was performed according to the method of Podlowski et al^[14] with minor modification. The slices were transferred to a microcentrifuge tube and subsequently homogenized by sonication in 120 µL of a cold PBS buffer consisting of phenathroline 1 mmol/L, iodacetamide 1 mmol/L, PMSF 0.4 mmol/L, pepstatin A 1 µmol/L. Protein concentrations were determined by nucleic acid and protein analyzer (BECKMAN DU 640) based on the Bradford method. Samples containing amounts of total protein (20 µg) were boiled for 5 min, separated on 12 % SDS-PAGE gel, transferred on PVDF membranes and blocked for 3 h at room temperature. Then the blots were incubated overnight with primary antibody. A rabbit polyclonal antibody (1:1000 Santa Cruz) was used as a primary antibody. An horseradish peroxidase-conjugated (1:1000) anti-rabbit Ig was used as a secondary antibody. Protein signals were visualized with enhanced chemiluminescence (Donpoint) and quantified with Gel Doc 1000 system (BIO-RAD).

Radioligand binding assay Rat hippocampal crude membrane was prepared according to the method based on Lai et al with modification^[15]. Hippocampal slice was homogenized with 20 fold volume buffer containing Tris-HCl 50 mmol/L, MgCl₂ 10 mmol/L, and then centrifuged. The concentration of protein was measured by nucleic acid and protein analyzer with Bradford method. ₁ Adrenergic receptor binding assay was carried out according to Stutz et al^[16]. Sample 50 µL was incubated with [³H]DHA at different concentration in a total volume of 250 µL for 30 min at 25 ºC. The incubation buffer consisted of Tris-HCl 50 mmol/L and MgCl₂ 10 mmol/L, pH 7.4. Non-labelled competitor propranolol was added to measure non-specific binding. After incubation, samples were filtered under vacuum over Whatman GF/G filter, and washed with ice-cold buffer. The amount of [³H]DHA was determined by LKB-1214-Rackbeta Counter at 60 % efficiency. B_max and K_d values were calculated by nonlinear regression analysis and normalized to the control.

Statistical assay The data were presented as mean±SD. Semiquantitative data of RT-PCR and Western blot were expressed as the percentage relative to controls in the same experiment. Statistical assay was performed using 2-Way ANOVA of SPSS software. P<0.05 was considered statistical significant.

RESULTS

RT-PCR RNA extracted from sample was scanned on the 1.5 % agarose gel with ethidium bromide staining, no degradation of RNA and contamination of DNA were found. The reverse transcription and amplification of total RNA without digestion isolated from hippocampcal slice resulted in a single band of the expected size of 188 bp using ₁-AR specific primer; No amplified products could be revealed in RNA preparation digested with RNase A, suggesting no contamination of DNA in the RNA samples. The amplified products were increased proportionally to the increase in the amount of template RNA and the number of cycles, indicating that the RT-PCR used in this study is sufficiently sensitive and accurate to detect changes in ₁-AR mRNA level. On the basis of similar titration, 28 cycles were adopted for measurements of ₁-AR mRNA and GAPDH mRNA (Fig 1).

Fig 1. (A) Representative picture of ethidium bromide staining of agarose gel for assessment of DNA-free RNA preparation from the hippocampal slice of rats. Lane 1: RNA samples were digested with RNase A; Lane 2: without reverse transcription before PCR; Lane 3-5: Normal RT-PCR with ₁-AR specific primer, GAPDH specific primer, and bis ₁-AR and GAPDH primers, respectively. Lane M: marker. (B) RT-PCR was performed using 3.0 µg total RNA with 18, 21, 24, 27, 30, and 35 cycles, respectively (Lane 1-6). (C) Lane1-5 with 28 cycles using 0.2, 0.4, 0.8, 1.6, and 3.2 µg total RNA respectively with 28 cycles.

Determination of ₁-AR mRNA in the hippocampal slice of rat No change in ₁-AR mRNA was found in the hippocampal slice without nicotine treatment (data not shown), but significant increase in ₁-AR mRNA level was demonstrated after nicotine 10 µmol/L treatment. The mRNA level of ₁-AR was increased significantly to 153 %±20 % of control after nicotine treatment for 60 min, but after 90-min and 120-min treatment, the mRNA level of ₁-AR was 136.8 %± 15.6 % and 128.2 %±17.7 % of control, respectively. Prolonged application of nicotine did not have further effects on ₁-AR mRNA expression (Fig 2).

Fig 2. (A) Expression of ₁-AR mRNA by RT-PCR in the hippocampal slice exposed to nicotine 10 µmol/L. Lane 1-5: Control and treatment of nicotine for 30, 60, 90, and 120 min. (B) ₁-AR mRNA level was normalized to GAPDH signal. n=6. Mean±SD. ^bP< 0.05 vs control (2-Way ANOVA of SPSS).

Effect of nicotine on ₁-AR protein level in hippocampal slice No signal was found in lane 1 because of no ₁-AR protein in loading sample. However, one band weight of 63 kDa appeared at the other lanes loaded with sample extracted from hippocampus, consistent to the other investigation^[9]. This findings indicated that the antibody was specific for ₁-AR. ₁-AR protein level in hipoocampal slice began to increase at 30 min. It was elevated by 54.3 % at 60 min (P<0.05) and reached peak level (by 81.5 %, P<0.01) at 90 min. It dropped at 120 min (67.4 % vs 81.5 %) (Fig 3).

Fig 3. Increase of ₁-AR protein level after treatment with nicotine 10 µmol/L. (A) Autograph of Western blot. Lane 1, negative control without ₁-AR protein, no band was revealed; Lane 2-6, 30 µg total protein extracted from hippocampal slice after nicotine 10 µmol/L treatment for 0, 30, 60, 90, and 120 min, respectively, only one band was revealed in each lane, the molecular weight is 63 kDa. Lane M: standard protein marker. (B) Western blot analysis of ₁-AR protein level was quantified with Doc Gel 1000 system. n=6. Mean±SD. ^bP<0.05 vs control (2-Way ANOVA of SPSS).

Effect of nicotine on maximum binding capacity of ₁-AR The maximal binding capacity (B_max) for [³H]DHA binding was greatly increased to 171.4 %± 17.3 % of control after treatment with nicotine 10 µmol/L for 60 min and reached to the peak (193.1 %±20.2 % of control) after nicotine treatment for 90 min. But when the period was prolonged to 120 min, B_max was 155.4 %±11.3 % of control (P<0.01 vs 90 min). The results reinforced the data that ₁-AR protein expression was up-regulated in the hippocampal slice after nicotine 10 µmol/L treatment. In contrast, no effect of nicotine on the affinity of ₁-AR was investigated (data not shown).

Fig 4. Effect of nicotine on the maximal binding capacity of ₁-AR in hippocampal slice using [³H]DHA. n=5. Mean±SD. ^bP< 0.05 vs control (2-Way ANOVA of SPSS). The density of control group is (67±16) pmol·g^-1protein.

DISCUSSION

Adrenergic system regulates forms of synaptic plasticity is involved in memory formation. LTP induced by nicotine is blocked by propranolol, a nonselective antagonist^[7,12], which indicated that -AR activation was involved in cognitive function improved by nicotine and other nicotinic receptor agonists. In present study, we directly observed that nicotine up-regulated ₁-AR mRNA and protein level.

Both ₁ and ₂ receptors are expressed in hippo-campus, but ₁-AR is predominant for cognitive function because it was expressed on neurons, while ₂-AR was expressed in blood vessels, thus we selected ₁-AR in present study. ₁-AR mRNA level and protein level were increased in hippocampcal slice after nicotine treatment. The fact that the peak of protein level appeared later than that of mRNA indicated the protein increase was referred to gene transcription. However, mRNA level was unchanged after treatment with nicotine for 30 min while the protein expression was markedly increased, which suggested there was at least a path way independent to gene transcription. So the change of protein level of ₁-AR induced by nicotine was due to not only the altered ₁-AR gene transcription but also regulation of post-transcription, such as post-transcriptional modification, translation, even degradation of protein. The effect of nicotine on ₁-AR gene post-transcription may be more significant. In this study, it should be noticed that the magnitude of changes in the ₁-AR density measured by radioligand assay is not corresponded with that determined by Western blot analysis, which may be due to the difference in the sensitivity of the two methods. Western blot is specific for ₁-AR but radioligand binding assay is not.

Induction of LTP is mediated by G protein signaling pathway. Activation of ₁-AR is one of the important components to G protein signaling pathway. ₁-AR agonist elicited increase in cAMP level and ionic concentration, which caused phosporylation of ERK^[17-19]. So the increase in ₁-AR mRNA and protein expression will improve cognitive function through G protein signaling pathway.

Interestingly, prolonged treatment with nicotine did not further up-regulated both mRNA and protein expression of ₁-AR. One of the possibility is that acute release of neurotransmitter induced by nicotine will up-regulate ₁-AR mRNA expression and protein synthesis, but lasting neurotransmitter release will inhibit up-regulation of ₁-AR mRNA transcription and protein synthesis^[20]. Another possibility is that nicotine receptor will desensitize after long-lasting stimulation by nicotine. Fu et al reported that nicotine receptor desensitized at 40 min after consecutive stimulation by high concentration of nicotine^[21].

In conclusion, nicotine induced LTP by up-regulating mRNA and protein expression of ₁-AR in hippocampcal slice.

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